1. Field of the Invention
This invention is directed to an article of manufacture and to a method. More specifically, this invention concerns itself with a test strip which can be used in a disposable fixture designed for the collection of a whole blood sample on the test strip. The test strip itself has two functional component elements, one component of which (wicking element) is intended for the collection and transport of the sample from the sample application site, and the second component thereof (porous membrane) is intended for the separation of interfering constituents from that portion of the sample subjected to analysis. This test strip is unique in that neither functional component is in contiguous relationship with the other, nor is fluid transport effected through an intermediate layer. The indicator used in the analysis system is capable of measurement by reflectance technique (spectroscopy).
2. Description of the Prior Art
The analysis of biological fluids to confirm the levels of various biological products is an accepted clinical practice for the determination of proper functioning of the endocrine system. This practice is common in the diagnosis of diabetes and in the management of this disease. Blood sugar levels can generally fluctuate with the time of day and with the period since the individual's last consumption of food. Management of diabetes often, thus, requires the frequent sampling and analysis of the diabetic's blood for determination of its relative glucose level. The management of this disease by the diabetic will typically involve the sampling of his own blood, the self-analysis of the sample for its relative glucose content and the administration of insulin, or the ingestion of sugar, depending upon the indicated glucose level. A number of devices have recently appeared on the market to assist the diabetic in the self-testing of the blood sugar level. One such device, developed by Audiobionics (now Garid, Inc.)--described in U.S. Pat. No. 4,627,445, issued Dec. 9, 1986. to be introduced into the marketplace in the not too distant future--involves the use of a fixture containing a multi-layered element for the collection of the whole blood sample, the transport of the sample from the point of application on the element to a porous membrane, and the analysis of the blood sample for its glucose contents by the dry chemistry reagent system which is present within the porous membrane.
In the preferred embodiments of this disposable fixture, the whole blood sample is obtained by a pin prick of the diabetic's finger, the blood collected on a defined area of a wicking element, and transported from the point of application on the wicking element to the porous membrane containing the dry chemistry reagent system. The configuration of the blood collection/dry chemistry reagent system within the fixture is preferably designed to insure the uniform delivery of the whole blood to the membrane containing the dry chemistry reagent system. In the preferred embodiments of this device, the colored components of the blood are effectively isolated, or separated from the blood sample by the membrane or a separate element within the reaction zone prior to the interaction of the serum component of the sample with the dry chemistry reagent system. If glucose is present in the sample, the chemistry reagent system will interact therewith, thus, forming an indicator which can be measured by a reflectance spectrophotometer of the device for which the fixture is adapted.
The subject matter of this invention is directed to an improvement in this multi-layered element which can be used in the fixture of the device described in the previous referenced co-pending patent application.
Preliminary to description of the improvements of this invention, it will be helpful to place it in context by review of the relevant prior art.
The performance of glucose determinations on whole blood, utilizing multiple layered films, is the subject of numerous publications and issued patents. The following listing is representative of the publications in this area: Dry Reagent Chemistries in Clinical Analysis, Analytical Chemistry, Vol. 55, No. 4, pp. 498-514 (April, 1983); Curme, Henry G., et al., Multilayer Film Elements for Clinical Analysis: General Concepts, Clinical Chemistry, Vol. 24, No. 8, pp. 1335-1342 (August, 1978); Spayd, Richard W., et al., Multilayer Film Elements for Clinical Analysis: Applications to Representative Chemical Determinations, Clinical Chemistry, Vol. 24, No. 8, pp. 1343-1350 (August, 1978); Ohkubo, Akiyuki, et al., Plasma Glucose Concentrations of Whole Blood, as Determined with a Multilayer-Film Analytical Element, Clinical Chemistry, Vol. 27, No. 7, pp. 1287-1290 (July, 1981); Ohkubo, Akiyuki, et al., Multilayer-Film Analysis for Urea Nitrogen in Blood, Serum, or Plasma, Clinical Chemistry, Vol. 30, No. 7, pp. 1222-1225 (July, 1984); and, Rupchock, Patricia, et al., Dry-Reagent Strips Used for Determination of Theophylline in Serum, Clinical Chemistry, Vol. 31, No. 5, pp. 737-740 (May, 1985). The following listing is representative of the patent literature in this area: U.S. Pat. Nos. 3,061,523 (to Free); 3,552,925 (to Fetter); 3,607,093 (to Stone); 4,042,335 (to Clement); 4,059,405 (to Sodickson, et al); 4,144,306 (to Figueras); 4,258,001 (to Pierce); and, 4,366,241 (to Tom, et al).
The above publications contain a relatively complete description of the state of the art which are embodied within devices which are available from Eastman Kodak Company and from the Ames Division of Miles Laboratories. U.S. Pat. No. 3,061,523 (to Free), describes the basic chemistry reagent system which has become the standard for colorimetric determination of glucose in biological samples. The chemistry system described by Free is contemplated for use in conjunction with a "dip stick" test. In a typical configuration of the Free invention, a solid phase (i.e. sticks or test strips) is pre-treated with his novel chemistry formulation. The reagent treated portion of this solid phase can thereafter be contacted with a sample suspected of containing glucose. The intensity of color which is developed as a result of such contact is compared to a control or standard and a semi-quantitative determination of glucose level in the sample thereby computed.
In the specific embodiments of the Free device, the dry chemistry reagent system is prepared by first dissolving the reactive constituents in a gelatin base and thereafter impregnating strips of filter paper with this dry chemistry reagent system. This is achieved by simply immersing the filter paper in the gelatin base/reagent system for a sufficient interval to effect impregnation of the reagents into the filter paper. The filter paper treated in this fashion is thereafter dried. The gelatin component of the impregnating solution is reportedly essential to the uniformity of color development. Presumably, the presence of the gelatin controls or inhibits the migration of fluids within the filter paper, thereby minimizing chromatographic separation of reagents and/or sample.
A drop of blood (preferably whole blood) is then applied to the portion of the filter paper containing the dry chemistry reagent system; allowed to react with the reagents contained therein (for approximately 60 seconds) and, thereafter the blood (presumably the red cells) rinsed from the paper. The intensity of the color indicator which is developed as a result of the interaction of the glucose and the reagents within the paper, is thereafter observed or measured. The recommendation (if not a requirement), of the Free system, that the red cells be rinsed from the surface of the test strip, implies that their removal is desirable, if not essential, to observation/measurement of the colored indicator.
Where the technician performing this test is dealing with a patient sample that may contain infectious microorganisms or viruses, the requirement that the sample be rinsed of red blood cells unnecessarily exposes the technician to potential infection.
U.S. Pat. No. 3,552,925 (to Fetter), represents an improvement to the glucose test element described in the Free patent. Fetter discloses a method and device for effectively separating the whole blood sample into its serum components and into its erythrocyte components (red blood cells and other color forming constituents). Fetter achieves this separation by treatment of a defined area of his sample collection device on his test element with certain water soluble salts. The contact of the whole blood sample with these salts in the test element results in the localized reaction of the erythrocytes (and the other colored components of the whole blood) with these salts with the resultant separation of the serum component therefrom. The serum fraction is, thus, free to migrate or diffuse into the test element. The migration and/or diffusion of the serum component is generally via capillary action or some other passive transport mechanism. The manner in which the sample is applied and the nature of the test medium, effectively transports and distributes the serum to another defined chemically reactive area of the test element containing test reagents. The test reagents of the test element are specific for one or more analytes of interest in the serum fraction (i.e. glucose, galactose, urea, uric acid, phenylalinine and/or various enzymes).
The various configurations of the Fetter test element contemplate a single laminae (FIGS. 1 and 8), having discrete areas of chemical treatments; or a multi-layered structure, wherein a single chemical treatment is confined to each of the layers of the laminate (FIGS. 3 through 7).
Fetter also indicates that the same matrix can be used to retain both the separating reagent and the reagent specific for the analyte of interest. In this latter embodiment of his invention, the whole blood sample would be applied to one side of the strip held in the horizontal position. After adequate penetration of the sample into the matrix containing both the separating and test reagents, the test strip would be inverted and color development observed (if any) on the site opposite the site of application of the whole blood sample. Fetter is not apparently concerned with potential interference of the colored blood components with the development and/or observation of the indicator species. It is, however, apparent that at low concentrations of analyte, the highly colored blood components would interfere and/or mask the presence of the indicator from visual observation/detection.
U.S. Pat. No. 3,607,093 (to Stone), describes the use of a filtration membrane as an analytical element for glucose analysis of whole blood. The membrane is pretreated with a chemical reagent system for glucose detection, and dried whole blood is applied to a dense surface of the membrane. The cellular components remain on top of the membrane, while the serum fraction is absorbed and reacts with the reagent system. The Stone configuration, as contemplated, is not readily adapted to instrument monitoring for the reasons given in the discussions of the shortcomings of the system described in the Free and Fetter patents.
U.S. Pat. No. 4,042,335 (to Clement), describes a multi-layered analytical element suitable for performing chemical analysis of whole blood samples. The Clement configurations all contemplate the application of test samples either directly, or from a spreading layer, to a reagent layer. The reagent layer contains a complement of chemicals for reaction with a specific analyte suspected of being present in the test sample. If the analyte is present, a "detectable species" is formed or released from the reagent layer and diffuses into what is termed a "registration layer"--that is, a layer whose sole function is to provide a medium or repository from which the detectable species can be observed or measured. In order to avoid interference (masking) in the observation or measurement of the detectable species, the registration layer is both devoid of the test sample and reagents used in the generation of the diffusable species. In the preferred embodiments of the Clement test element, an optical screen ("radiation blocking layer") is also provided between the reagent layer and the registration layer. This optical screen effectively optically isolates the detectable species from other constituents which could interfere in its detection and/or measurement.
As is evident from the foregoing description, Clement attempts to segregate the individual functions of his analytical element into discrete layers. This technique, although potentially attractive to a manufacturer in possession of technology for fabrication of multi-layered elements, is by its very nature unduly complex and potentially prone to mechanical instability of the composites. More specifically, where this test element is to be used by an individual in a self-test environment, the composite must necessarily be supported on an additional element to lend physical integrity to the multi-layered element and thereby prevent its unintended flexing and potential separation of the various layers contained therein.
U.S. Pat. No. 4,059,405 (to Sodickson, et al), describes a method and apparatus for glucose analysis of whole blood samples. In the Sodickson system (as described in Example I d.), a reaction site is initially prepared by preforming wells in a polyox resin treated filter paper. A reaction site is physically defined in this treated paper by impressing thereon a confining ring approximately one centimeter in diameter. A glucose reagent is then applied to the reaction site defined by this ring and the reaction site dried. An ultrafiltration membrane is placed over the well and a sheet of paper containing a dried blood spot placed in contiguous relationship with the ultrafiltration membrane. The dried blood spot is then reconstituted by the addition of saline. The apparatus used in the Sodickson system (i.e. press) confines the reconstituted blood sample in the reaction well for a brief period. During this incubation period, soluble components of the whole blood sample are redissolved in the saline and pass through the ultrafiltration membrane where they come in contact with the glucose test reagents in the polyox treated paper. The cellular components of the blood are retained on the ultrafiltration membrane and thereby prevented from interference and measurement of the glucose manifesting indicator.
The system described by Sodickson, as contemplated in his Example I d., is cumbersome (requiring reconstitution of the blood sample and relatively complex equipment to effect separation of cellular components from the whole blood sample) and does not readily lend itself to self-testing.
U.S. Pat. No. 4,144,306 (to Figueras), describes a multi-layered analytical element analogous to that of the Clement patent (previously discussed). The Figueras chemistry differs from Clement in that the interaction of an analyte and non-diffusable reagents in the reagent layer, results in the release of a "preformed detectable species" which can migrate from the reagent layer into a registration layer. This performed detectable species is then observed or measured in the registration layer. Figueras contemplates (as described in Example VI) the adaptation of his system to glucose analysis of whole blood. The separation of colored and cellular components of the whole blood would be achieved by Figueras in essentially the same fashion as in the Clement patent. The introduction of the whole blood sample into the reagent layer of the Figueras element results in the release of a diffusable preformed photographic dye, which is then free to migrate into the registration layer. Figueras requires the presence of the same type of optical screen (radiation blocking layer) between the reagent layer and the registration layer to avoid masking or interference in detection of the dye from the nondiffusable color components (i.e. sample and reagents) in the reagent layer. The limitations and disadvantages noted in the discussion of the Clement patent are also applicable to the multi-layered analytical element of Figueras. Figueras, however, introduces an initial complexity; namely, the effective immobilization of the reagents within the reagent layer and the preservation of the preformed indicator prior to its release by the analyte of interest. Because of the requirements of maintenance of fluid contact between the various elements of the Figueras composite, its mechanical properties are critical. Accordingly, the multi-layered element of Figueras, as previously noted for Clement, will require a supporting (transparent) layer to lend physical integrity to this device.
U.S. Pat. No. 4,258,001 (to Pierce et al), describes a multi-layered analytical element (of the type described in both Clement and Figueras--previously discussed) incorporating a unique spreading layer. The spreading layer of the Pierce patent is described as an essentially "non-fibrous" material. In one of the preferred embodiments described by Pierce (FIG. III), the spreading layer can contain "interactive compositions" (test reagents) for reaction with analytes in a test sample. Pierce also contemplates the use of her device in the analysis of whole blood, blood serum and urine. Whole blood can be applied directly to the Pierce element. The presence of red blood cells will not reportedly interfere with spectrophotometric analysis if carried out by reflectance measurements, provided a radiation screen (blocking layer) is used to screen out interference from the red cells (column 26, line 49-61).
As is evident from this patent, the Pierce device is designed to "take up" the whole blood sample. Thus, both cellular and non-cellular components of the whole blood are imbibed by the spreading layer. The spreading layer of Pierce is, thus, not intended nor contemplated as a means for separation of the cellular fraction of the blood from the serum fraction. Where enzyme based diagnostic clinical assays are incorporated into the spreading layer (as in the case of glucose analysis), the potential for inhibition of these enzymes by the erythrocytes can potentially mask low concentrations of glucose and, thus, distort an otherwise clinically significant result.
The transport and spreading of biological samples is of concern, not only in clinical chemistry system of the type described in the above patents, but also in immunoassays of the same biological fluids. The following U.S. Patents are representative of the literature in this area: U.S. Pat. No. 4,094,647 (to Deutsch); and U.S. Pat. No. 4,366,214 (to Tom et al).
U.S. Pat. No. 4,094,647 (to Deutsch), describes a linear wick having defined areas for placement of reagents and sample. The end of the wick is placed in a vertical position as a developer fluid and the fluid drawn up the wick by capillary action. As the fluid is drawn into the wick, it transports the reagents and sample, from their respective locations, into contact with one another. One or more of these reagents can be immobilized on the wick; thus, the developer fluid is used to transport reagent and sample to the immobilized reagent and any unreacted or mobile materials from the immobilized reagent. The site having the immobilized reagent can then be viewed or measured for the presence of analyte.
U.S. Pat. No. 4,366,241 (to Tom et al) describes a device for the non-chromatographic immunoassay of biological fluids. In the Tom assay device configuration, a test element, having a relatively small test zone, is treated with an immobilized binding material (termed "mip" or "member of an immunological binding pair"). The test zone of this device is the exclusive entry port for the biological sample and is designed for receipt of the biological sample either by direct application or immersion in the test fluid. The analyte (if any) contained in the biological sample is selectively (immunochemically) bound in the test zone to the immobilized binding material which is specific for this analyte. The residual components of the sample including the fluid component thereof, are drawn from the test zone to a second element which is in fluid contact (contiguous relationship) with the test zone. The second element's function is to pump or draw the biological sample through the test zone into the test element. Those constituents of the sample which are not bound in the test zone are, thus, drawn into the test element and away from the test zone.
In the immunological test element of the type described by Tom, the pretreatment of the test zone effectively confines the analyte and the test reagents (i.e. labelled indicator) to the analysis site, thereby effectively eliminating the problems of reagent and sample migration which are common in the solid phase systems designed for clinical chemistry analysis. These immunoassay systems of Tom are not, however, without their disadvantages, the most common being the nonspecific binding of interfering substances in the reaction zone and the difficulties which are sometimes inherent in the detection of low levels of analyte.
As is evident from the foregoing discussion of the references suitable for whole blood analysis, each type of element generally requires a plurality of lamina in its preferred configuration. Where a single layer (component) test device is suggested, none of the references, with one exception (U.S. Pat. No. 3,607,093-to Stone), either acknowledge or appreciate the potential chemical and optical interference of the erythrocyte population (and other colored components of the blood), on the analytical protocol or in the detection of the reaction product which is indicative of the presence of the analyte (namely, the glucose). Where immobilization techniques (as described in Fetter, Deutsch and Tom) are employed, the specificity of the binding reaction can, under certain conditions, be indiscriminate. In the case of Fetter, the attempt at scavenging of erythrocytes and other colored components from the sample with certain salts that have been imbibed within the test medium, is not without its limitations. In the single layered element of Fetter, the serum fraction is readily radially separated from the colored component of the blood and thereby results in the distribution of the analyte over a relatively large area. If the analyte is present in low concentrations, it can easily escape detection. Thus, some amplification mechanism may be required for visualization of a low level of analyte (the use of an enzyme in conjunction with a chromogenic substrate).
The immunochemical device and techniques of the type described by Deutsch and Tom are not readily compatible with whole blood analysis. It is possible to wash the test cell for removal of the cellular debris (as is suggested in the Free patent), however, the effect of such additional step upon the immunochemical binding process is not known and exposes the clinician or the person performing the test to potential infection by those components of the blood which are removed from the test element.
In summary, it should now be apparent that the prior art lacks a simple yet accurate device for glucose analysis of whole blood. Where such devices have been proposed, they are complex, do not readily lend themselves to self-testing without the provision of a fixture or additional support mechanism, and generally lack the sensitivity to permit differentiation of different levels of analyte over a broad clinical range of concentration. Moreover, all such devices discussed above (with the exception of the Stone patent) would appear to have one common failing; namely, their inability to effect optical (and in certain instances, chemical) isolation of erythrocyte and other colored components of the whole blood from the observation/measurement site without the physical removal of the erythrocytes from the test element; or, the provision of some optical screen (blocking layer) between the colored components of the sample and the indicator compound which is generated as a result of the clinical assay.